Malaria is affecting 40% of the world's population with an estimated 1.5-2.7 million deaths annually (32). This represents a tremendous human suffering and a burden that prevents the development of the affected endemic communities. Malaria is now almost confined to the poorest tropical areas of Africa, Asia and Latin America, but transmission is being reintroduced to areas where it had previously been eradicated. Malaria is one of the world's greatest public health problems.
The increasing emerging of insecticide resistant vectors and drug resistant parasites calls for investment in new and better control tools. Malaria vaccines hold the potential to dramatically alleviate the burden of malaria. However, our understanding of the mechanisms underlying protective immunity is incomplete hence specific markers of protection still needs to be defined.
An effective malaria vaccine will require the induction of appropriate humoral and cellular immune responses, against several key parasite antigens expressed during the various stages of the parasite life cycle. Each stage in the life cycle provides an opportunity for a vaccine.
Presently, three main lines of malaria vaccine research dominate: (i) induction of immunity against pre-erythrocytic antigens, a strategy rooted in first experiments with UV-inactivated P. gallinaceum sporozoites (25), (ii) identification of antigens that induce antibodies with specificities similar to those of immunoglobulin preparations of semi-immune adults with a therapeutic effect in malaria patients (5), and (iii) induction of transmission-blocking (TB) antibodies against parasite antigens that are expressed in the infected mosquito (11). The first two strategies rely on malaria antigens that induce a protective immune response, and the third strategy on malaria antigens that are essential for sexual development of the parasites in the infected mosquito.
The objective of a transmission-blocking malaria vaccine (TBMV) is to prevent an individual from becoming infected with Plasmodium parasites by mosquito bites of the Anopheles vector. As a result, the spread of malaria in the population is expected to decrease with subsequent reduction of the disease. TBMVs are based on sexual- or sporogonic-specific antigens and designed to arrest the development of sporogonic stages inside the mosquito. The specific antibodies generated in the human host are passively ingested together with parasites when mosquitoes take a blood meal and will bind to the parasites thereby preventing progression of their sporogonic development. Once inside the mosquito midgut, gametocytes rapidly emerge from the intracellular red blood cell environment to prepare for fertilization and are directly exposed to hostile immune components of the ingested blood. The sporogonic cycle is biologically the most vulnerable part of the lifecycle because parasite numbers are very low which makes this an attractive target for interventions.
The Plasmodium falciparum Pfs48/45 is a sexual stage-specific protein expressed by gametocytes (2, 12) and present on the surface of the sporogonic (macrogametes) stages of the malaria parasites. Pfs48/45 plays a key role in male gamete fertility and zygote formation e.g. parasite fertilization (29) and antibodies which target conformational epitopes of Pfs48/45 prevent fertilization (22, 31). Specific antibodies against Pfs48/45 are present in human sera from endemic areas (23) and correlate with TB activity (4, 23-24, 27).
Five distinct B-cell epitopes (epitope II is subdivided into IIa and IIb) have been defined based on binding studies with a panel of Pfs48/45 specific monoclonal antibodies (24) (FIG. 1). Epitopes I-III in the C-terminal domain of the protein are conformational and epitope IV is linear. For epitope V in the N-terminal domain, both linear- and conformation-dependent monoclonal antibodies have been described (24). Monoclonal antibodies to epitope I and V block transmission effectively in the membrane feeding assay but monoclonal antibodies of epitope IIb and epitope III were ineffective on their own but able to reduce transmission when used in combination (3, 21, 30).
Pfs48/45 has been produced on recombinant form in different expression systems; however, the major challenges with recombinant Pfs48/45 are that it is very difficult to produce correctly folded protein. Proper folding of many CYRPs, including Pfs48/45, depends on correct formation of disulphide bridges. In eukaryotes the oxidizing environment of the endoplasmic reticulum (ER) provides a milieu for disulphide bonds formation. Prokaryotic organisms such as Escherichia coli and Lactococcus lactis lack the sophisticated ER machinery of disulphide bond formation. In Escherichia coli correct disulphide bonds are formed in the periplasmic space catalyzed by a set of periplasmic oxidoreductases. Accordingly, the C-terminal Pfs48/45 fragment (10C) (FIG. 1) was produced as a correctly folded protein in the periplasm of Escherichia coli when genetically fused to the maltose binding protein (MBP) and co-expressed with four periplasmic folding catalysts, (17). Levels of up to 1 mg/L pure correctly folded material was reported. Such expression levels are insufficient for further up-scaling and GMP production.
It is therefore, desirable to develop a large scale production method for a vaccine based on a recombinant protein, which include Pfs48/45 or other cysteine-rich antigens from P. falciparum such as the Pfs25, Pfs47, Pfs230, EBA175 and Var2CSA antigens.